Download Cloning vectors

Cloning vectors
•Propagation of DNA
–gene of interest
–cDNA or genomic library
•Manipulation of DNA
–nucleotide sequencing
–site-directed mutagenesis
•Delivery of DNA
–expression of large quantities of protein
–functional expression
Types of cloning vectors
• plasmids
• filamentous phages
• lambda phages
• cosmids
• bacterial artificial chromosomes (BACs)
• yeast artificial chromosomes (YACs)
Plasmids
Plasmids are double-stranded,
circular, self-replicating, extrachromosomal DNA molecules.
Plasmids (continued)
• a replicator
– ColE1, pMB1
– copy-number
• a selectable marker
– dominant (e.g. Amp, Tet, Kan)
– recessive (e.g. LeuB)
• a multiple cloning site (MCS)
– restriction sites
Plasmids (continued)
• 8-bp-cutter sites for insert excission
• oligonucleotide complementary sequences
• RNA promoters for transcription of cRNA
• LacZ gene for insert selection
• Trc promoter for regulated expression in E. coli
• propagation, selection and expression outside E. coli
• transcriptional / translational fusion products
Philamentous phages
Philamentous phages are singlestranded DNA phages that infect F-factor
containing E. coli, and can be recovered
in both single-stranded (phage) and
double-stranded (plasmid) form.
Philamentous phages (continued)
• examples: f1, M13, fd
• advantages: very useful for sequencing
and mutagenesis
• some plasmids can propagate as
“phagemids” when co-transfected with
helper phages
Lambda phages
Lambda phages are ~50 kb doublestranded DNA phages that replicate
in E. coli either in the lytic or in the
lysogenic mode.
Lambda phages (continued)
• examples: EMBL3, λgt10, λZAP
• suitable for either small (0-5 kb) or large
(10-20 kb) inserts
• efficient cloning and easy maintanace
(cDNA and genomic libraries)
• easy screening of large amounts of
recombinant clones (libraries)
wild-type lambda DNA
λ ZAP
λ DASH
Cosmids, BACs and YACs
These vectors can host extremely
large inserts (upto 50 kb for cosmids,
and 100-500 kb for BACs and YACs),
and are primarily used in genome
sequencing projects.
What determines the choice vector?
• insert size
• vector size
• restriction sites
• copy number
• cloning efficiency
• ability to screen for inserts
• what down-stream experiments do you plan?